Retinitis pigmentosa (RP) is a group of diseases in which one of hundreds of different mutations leads to death of rods resulting in night blindness followed by gradual death of cones causing constriction of visual fields and eventual blindness. The loss of rods results in elevated levels of oxygen in the outer retina and during the current grant period, we demonstrated that this is accompanied by progressive oxidative damage in cones causing reduced function and cell death. Using a transgenic approach, we demonstrated that over-expression of components of the endogenous antioxidant defense system in mouse models of RP can reduce oxidative damage and promote cone function and survival, but only if there is co- expression of a superoxide dismutase (SOD) and a H2O2-detoxifying enzyme in mitochondria or in the cytosol. This suggests that in the setting of RP, there are excessive reactive oxygen species (ROS) in both the cytosol and mitochondria and also indicates that in order to utilize ROS-detoxifying enzymes to treat RP, multi- gene transfer will be needed. We began translating our findings into a gene-based therapy for RP by identifying a viral vector/promoter combination that transduces photoreceptors after subretinal or intravitreous injections in mice or pigs and we initiated studies with therapeutic transgenes in mouse and pig models of RP. During the next grant period we use quantitative techniques to assess the effect of multi- gene transfer on the expression of individual component transgenes. We will continue our current experiments in both mouse and pig models of RP that will answer whether targeting the mitochondria or cytosol with multi-gene transfer provides greater cone rescue. Finally we will determine whether targeting both the mitochondrial and cytosolic compartments provides better outcomes than targeting either alone. The potential impact of this proposal is high because it will provide important new information regarding multi-gene transfer that will interest all investigator in the gene therapy field, and most importantly will provide critical efficacy data in a large animl model of RP that could lead to clinical trials.